Copy number variations are not modifiers of phenotypic expression in a pair of identical twins carrying a BRCA1 mutation.

Mutations in BRCA1 and BRCA2 genes confer a high risk of breast and ovarian cancer but the incomplete penetrance of these mutations suggests that other genetic and/or environmental factors may modify this risk. We present a family where all affected members carried a mutation in the BRCA1 gene and the index case had suffered from cancer twice in the last 27 years, whereas her monozygotic twin sister, also a carrier of the mutation, remained healthy. As copy number variants (CNVs) contribute to phenotypic diversity, a comparative genomic hybridization array (CGH) was performed to see whether the differences in the CNV profile were a modifier factor of the phenotype in our monozygotic twins. Our results show that differences in the CNVs profile were not the cause of the extremely variable penetrance observed in our MZ twin. The search for an explanation should not therefore be limited to genetic changes at the level of the DNA sequence.

Mutational Screening of BRCA1/2 Genes as a Predictive Factor for Therapeutic Response in Epithelial Ovarian Cancer: A Consensus Guide from the Spanish Society of Pathology (SEAP-IAP) and the Spanish Society of Human Genetics (AEGH).

Germline/somatic BRCA-mutated ovarian carcinomas (OC) are associated to have better response with platinum-based chemotherapy and long-term prognosis than non-BRCA-associated OCs. In addition, these mutations are predictive factors to response to Poly(ADP-ribose) polymerase (PARP) inhibitors. Different positioning papers have addressed the clinical recommendations for BRCA testing in OC. This consensus guide represents a collection of technical recommendations to address the detection of BRCA1/2 mutations in the molecular diagnostic testing strategy for OC. Under the coordination of Spanish Society of Pathology (SEAP-IAP) and the Spanish Society of Human Genetics (AEGH), these recommendations have been developed by pathologists and geneticists taking into account previously published recommendations and their experience in the molecular characterization of these genes. Since the implementation of BRCA testing as a predictive factor can initiate the workflow by testing germline mutations in the blood or by testing both germline and somatic mutations in tumor tissue, distinctive features of both strategies are discussed. Additionally, the recommendations included in this paper provide some references, quality parameters, and genomic tools aimed to standardize and facilitate the clinical genomic diagnosis of OC.

p53 regulates the self-renewal and differentiation of neural precursors.

During embryo neurogenesis, neurons that originate from stem cells located in the forebrain subventricular zone (SVZ) continuously migrate to the olfactory bulb (OB). However, other authors describe the occurrence of resident stem cells in the OB. In the present work we report that the absence of tumor suppressor protein p53 increases the number of neurosphere-forming cells and the proliferation of stem cells derived from 13.5-day embryo OB. Interestingly, differentiation of p53 knockout-derived neurospheres was biased toward neuronal precursors, suggesting a role for p53 in the differentiation process. Moreover, we demonstrate the relevance of p53 in maintaining chromosomal stability in response to genotoxic insult. Finally, our data show that neurosphere stem cells are highly resistant to long-term epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) deprivation in a p53-independent fashion, and they preserve their differentiation potential. Thus, these data demonstrate that p53 controls the proliferation, chromosomal stability and differentiation pattern of embryonic mouse olfactory bulb stem cells.

Normal telomere length and chromosomal end capping in poly(ADP-ribose) polymerase-deficient mice and primary cells despite increased chromosomal instability.

Poly(ADP-ribose) polymerase (PARP)-1, a detector of single-strand breaks, plays a key role in the cellular response to DNA damage. PARP-1-deficient mice are hypersensitive to genotoxic agents and display genomic instability due to a DNA repair defect in the base excision repair pathway. A previous report suggested that PARP-1-deficient mice also had a severe telomeric dysfunction consisting of telomere shortening and increased end-to-end fusions (d'Adda di Fagagna, F., M.P. Hande, W.-M. Tong, P.M. Lansdorp, Z.-Q. Wang, and S.P. Jackson. 1999. NAT: Genet. 23:76-80). In contrast to that, and using a panoply of techniques, including quantitative telomeric (Q)-FISH, we did not find significant differences in telomere length between wild-type and PARP-1(-/)- littermate mice or PARP-1(-/)- primary cells. Similarly, there were no differences in the length of the G-strand overhang. Q-FISH and spectral karyotyping analyses of primary PARP-1(-/)- cells showed a frequency of 2 end-to-end fusions per 100 metaphases, much lower than that described previously (d'Adda di Fagagna et al., 1999). This low frequency of end-to-end fusions in PARP-1(-/)- primary cells is accordant with the absence of severe proliferative defects in PARP-1(-/)- mice. The results presented here indicate that PARP-1 does not play a major role in regulating telomere length or in telomeric end capping, and the chromosomal instability of PARP-1(-/)- primary cells can be explained by the repair defect associated to PARP-1 deficiency. Finally, no interaction between PARP-1 and the telomerase reverse transcriptase subunit, Tert, was found using the two-hybrid assay.

Detection of known and novel genomic rearrangements by array based comparative genomic hybridisation: deletion of ZNF533 and duplication of CHARGE syndrome genes.

BACKGROUND: Mental retardation can be caused by copy number variations (deletions, insertions, duplications), ranging in size from 1 kb to several megabases. Array based comparative genomic hybridisation (array-CGH) allows detection of an increasing number of genomic alterations. METHODS: A series of 46 patients with mental retardation and congenital abnormalities (previously screened for subtelomeric rearrangements) were evaluated for cryptic chromosomal imbalances by array-CGH. This array contains 6465 large-insert BAC/PAC clones, representing sequences uniformly distributed throughout the human genome. The results were confirmed by alternative techniques. RESULTS: Four pathogenic rearrangements were detected: two of them were novel, a deletion at 2q31.2 and a duplication at 8q12 band; the other two have been previously reported--a duplication of the Williams-Beuren region and a deletion of 3q29. By adding the subtelomeric alterations previously identified, a total rate of 18% of pathogenic rearrangements was found in the series. CONCLUSION: Based on our results, ZNF533 is the only gene contained in the overlapping region with other deletions at 2q31.2, and it is most probably the fourth zinc-finger gene implied in mental retardation. On the other hand, we propose that the CHD7 gene, associated with CHARGE syndrome by haploinsufficiency, causes a different phenotype by gain-of-dosage.

Severe and recurrent episodes of bronchiolitis obliterans organising pneumonia associated with indolent CD4+ CD8+ T-cell leukaemia.

The present study describes an adult male who has had recurrent episodes of pulmonary infiltrates with severe acute respiratory failure over a period of 10 yrs. Clinical and pathological characteristics revealed bronchiolitis obliterans with organising pneumonia (BOOP) that responded dramatically to prednisone. BOOP is characterised by inflammation of the bronchioles and surrounding tissue in the lungs. It can mimic infectious pneumonia but diagnosis is suspected when there is no response to multiple antibiotic treatment, and blood and sputum cultures are negative for microorganisms. A high proportion of double-positive (DP)-T-cells was detected in peripheral blood and in bronchoalveolar lavage, expressing CD4 and CD8alphabeta heterodimer with memory phenotype. These DP-T-lymphocytes expressed specific homing molecules that could explain their tropism to lung tissue, giving rise to the clinical symptoms. The patient did not present organomegaly, lymphadenopathy, lymphocytosis or other features of malignancy. However, T-cell receptor Vbeta chain analysis indicated clonal rearrangement, and cytogenetic studies displayed chromosomic alterations that were similar to clonal proliferation observed in ataxia-telangiectasia and T-prolymphocytic leukaemia. The findings suggest a smouldering form of lymphoproliferation, the first sign of which was bronchiolitis obliterans organising pneumonia requiring constant corticoid treatment.

DNA profiling analysis of 100 consecutive de novo acute myeloid leukemia cases reveals patterns of genomic instability that affect all cytogenetic risk groups.

We have carried out a high-resolution whole genome DNA profiling analysis on 100 bone marrow samples from a consecutive series of de novo acute myeloid leukemia (AML) cases. After discarding copy number changes that are known to be genetic polymorphisms, we found that genomic aberrations (GA) in the form of gains or losses of genetic material were present in 74% of the samples, with a median of 2 GA per case (range 0-35). In addition to the cytogenetically detected aberration, GA were present in cases from all cytogenetic prognostic groups: 79% in the favorable group, 60% in the intermediate group (including 59% of cases with normal karyotype) and 83% in the adverse group. Five aberrant deleted regions were recurrently associated with cases with a highly aberrant genome (e.g., a 1.5 Mb deletion at 17q11.2 and a 750 kb deletion at 5q31.1). Different degrees of genomic instability showed a statistically significant impact on survival curves, even within the normal karyotype cases. This association was independent of other clinical and genetic parameters. Our study provides, for the first time, a detailed picture of the nature and frequency of DNA copy number aberrations in de novo AML.

Molecular cytogenetics in translational oncology: when chromosomes meet genomics.

The discovery of the genetic changes that contribute to cellular neoplastic transformation is one of the major aims in oncological research. Chromosome rearrangements account for a large part of these initiating mutations that, resulting in gene deregulation, are the main target of molecular cytogenetics. Cytogenetics, based in reasoned genomic and biological questions and supported by the development of new biotechnological tools, is a powerful discipline that is continuously generating pieces of information that have immediate translation as reagents for diagnosis and useful research data. The present review presents a summary of the major cytogenetic findings that already have a clear role in clinical oncology because of their use as diagnostic markers, as indicators of molecular therapy suitability or both. We also present an updated description of the molecular cytogenetics tools that have included genomic advances in their most recent releases: multicolour fluorescence in situ hybridisation methods (e.g. SKY karyotyping) and array-based comparative genomic hybridisation (arrayCGH).

DNA profiling by arrayCGH in acute myeloid leukemia and myelodysplastic syndromes.

Acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) represent two distinct but related myeloid haematological neoplasms. At diagnosis, a substantial proportion of cases show cytogenetic and molecular genetic markers whose range of specificity is highly variable. Most specific reciprocal translocations, as t(8;21)(q21;q21) or t(15;17)(q22;q21), have been extensively studied and are currently introduced in clinical diagnosis. Two other major groups remain to be better characterized at the genetic and genomic level: cases with normal karyotype and cases with complex aberrations. Comparative genomic hybridization (CGH) performed on chromosomes was the first approach taken and nearly 300 cases studied by this technique have already been reported. Array based CGH has also been applied to a smaller number of cases. Both types of genomic studies have confirmed that recurrent genomic losses and gains can almost exclusively be found in cases with complex karyotype. In most cases with normal karyotype (as well as in others with single chromosome aberrations as trisomy 8), arrayCGH has been able to unveil small DNA copy number changes whose recurrence is very low. Recently, single- nucleotide-polymorphism based arrays have been used in AML showing that loss of heterozygosity (LOH) is a common feature in normal karyotype leukemia.

Truncated RUNX1 protein generated by a novel t(1;21)(p32;q22) chromosomal translocation impairs the proliferation and differentiation of human hematopoietic progenitors.

We have identified a new t(1;21)(p32;q22) chromosomal translocation in a MDS/AML patient that results in expression of an aberrant C-terminally truncated RUNX1 protein lacking several regulatory domains. As similar truncated RUNX1 proteins are generated by genetic aberrations including chromosomal translocations and point mutations, we used the t(1;21)(p32;q22) chromosomal translocation as a model to explore whether C-terminally truncated RUNX1 proteins trigger effects similar to those induced by well-characterized leukemogenic RUNX1 fusion genes. In vitro analysis of transduced human hematopoietic/progenitor stem cells showed that truncated RUNX1 proteins increase proliferation and self-renewal and disrupt the differentiation program by interfering with RUNX1b. These effects are similar to but milder than those induced by the RUNX1/ETO fusion protein. GSEA analysis confirmed similar altered gene expression patterns in the truncated RUNX1 and RUNX1/ETO models, with both models showing alterations in genes involved in self-renewal and leukemogenesis, including homeobox genes, primitive erythroid genes and leukemogenic transcription factors. We propose that C-terminally truncated RUNX1 proteins can contribute to leukemogenesis in a similar way to RUNX1 fusion genes but through a milder phenotype.

Simultaneous detection of the immunophenotypic markers and genetic aberrations on routinely processed paraffin sections of lymphoma samples by means of the FICTION technique.

Disciplines such as morphology, immunophenotyping and genetics widely contributed over decades to the understanding of the cellular mechanisms of cancer. To obtain a greater insight into the complex processes of tumorigenesis, scientists have joined their efforts to combine many of the available techniques. Here, we report on the development of a FICTION (Fluorescence Immunophenotyping and Interphase Cytogenetics as a tool for the Investigation of Neoplasms) technique that allows a simultaneous detection of immunophenotypic markers and genetic aberrations on routinely processed lymphoma samples. As the antigen retrieval method seems to play an important role in the final results, we tested the pressure-cooking method at different times (2, 4 and 8 min) using three different buffers (EDTA, Tris-EDTA and citrate), resulting in improved sensitivity for the detection of both immunophenotypic markers and genetic aberrations. We also applied this method to different types of lymphoma using double immunofluorescence assays (including CD30, CD20, CD8 monoclonal antibodies) and several fluorescence in situ hybridization probes to demonstrate that the FICTION technique could be easily applied on paraffin sections in different combinations for the diagnosis and research of cancer.

Chromosomal changes pattern and gene amplification in T cell non-Hodgkin's lymphomas.

T cell non-Hodgkin's lymphomas are a heterogeneous group of lymphomas with poor prognosis, and whose genetic alterations are not well understood. Comparative genomic hybridization (CGH) is a technique that allows the identification of DNA imbalances without cytogenetic studies. We have studied 37 samples from 29 T cell non-Hodgkin's lymphomas (25 peripheral and four lymphoblastic lymphomas) by CGH in order to detect DNA sequence copy number changes of putative importance in the biology and prognosis of these neoplasms. We detected abnormal CGH profiles in 16/27 (59%) of samples at diagnosis, a ratio that increased to 66% (23/37) when we included the relapsed samples. The most common recurrent changes were gains related to the X chromosome, either the whole chromosome or partially the Xq26-27 bands (19%). Other recurrent changes included gains of bands 9q34, gains of chromosomes 17, 19, and 20, and complete or partial deletions of chromosome 13 (10%). Cancer-related genes located at Xq26-28 region were analyzed by Southern blot and fluorescence in situ hybridization (FISH). Low level amplification of some of these genes was detected by this technique confirming the results obtained by CGH in this region. The detection of abnormal CGH profiles in these T cell lymphomas could have clinical implications. Patients with abnormal CGH profiles showed significant associations with advanced stage of disease, overexpression of P53, and higher proliferative index.

De novo trisomy 16p.

We report on a patient with psychomotor retardation and a pattern of malformations comprising single umbilical artery, craniofacial anomalies, severe truncal hypotonia, and lower-limb hyporreflexia. G-banding cytogenetics demonstrated a 16p+ chromosome. Parental chromosomes were normal. The use of fluorescent in situ hybridization (FISH) showed that this extra material derived from chromosome 16. High-resolution G-banding demonstrated a duplicated segment on the 16p arm, confirming our suspicion of a de novo tandem duplication; hence, the cytogenetic diagnosis was given as 46,XY,dir dup(16)(p11.2-->p12).

Screening for genetic aberrations in papillary thyroid cancer by using comparative genomic hybridization.

BACKGROUND: Determination of the genetic composition of papillary thyroid cancers may help explain differences in observed clinical behavior. Comparative genomic hybridization (CGH) is a novel molecular cytogenetic assay that allows simultaneous detection of gains, losses, and amplification of genetic information, making it an ideal screening tool. The aim of this study was to identify genetic aberrations occurring in papillary thyroid cancers by using CGH analysis. METHODS: CGH analysis was performed on 21 individual cases of papillary thyroid cancers. Nonparametric statistical comparisons were performed with the Fisher exact test. RESULTS: Genetic abnormalities were identified by CGH in 10 of 21 cases (48%). A recurrent pattern of aberrations was seen in cases where genetic changes were detected, involving losses at chromosome arms 1p and 9q and chromosomes 17, 19, and 22, and gains at chromosome 4 and chromosome arms 5q, 6q, 9q, and 13q. The loss of chromosome 22 was unique to younger patients (P =.05) and was associated with a higher rate of regional lymphatic metastasis (19% vs 80%, P =.02). CONCLUSIONS: Two genetically unique groups of patients were identified by using CGH analysis. One group had no detectable aberrations; the other had a recurrent pattern of aberrations, localizing to the identical chromosomal loci. This pattern of aberrations suggests that the involved loci may contain genes important in thyroid carcinogenesis. The clinical significance of the presence of copy number changes detected by CGH needs to be determined. In addition, molecular cloning of involved genes in each of the aberrations is warranted.

A recurrent translocation, t(3;11)(q21;q13), found in two distinct cases of acute myeloid leukemia.

We report two cases of acute myeloid leukemia (M1 and M5B subtypes) with a similar translocation, t(3;11)(q21;q13). We discuss the involvement of these breakpoints in acute leukemia and their putative clinical implications.

Complex karyotype including 14q+ marker in a case of Waldenström's macroglobulinemia.

We describe a case of Waldenström's macroglobulinemia with a complex karyotype including a 14q+ marker. Secondary changes affected chromosomes 2, 4, 6, 7, 8, and 17. The cytogenetic significance of the changes and their prognostic value, as compared with those described in previous reports, are discussed.

A variant t(14;17) in acute promyelocytic leukemia. Positive response to retinoic acid treatment.

We present a case of acute promyelocytic leukemia (APL) carrying an atypical translocation involving chromosomes 14 and 17. This translocation could be considered a variant of the APL-specific t(15;17). Positive response to retinoic acid treatment suggests molecular rearrangement of retinoic acid receptor alpha.

Complex cytogenetic abnormalities including telomeric associations and MEN1 mutation in a pediatric ependymoma.

Ependymomas are neuroectodermal tumors of the brain and spinal cord. Some recurrent cytogenetic aberrations have been reported in these tumors, including alterations involving chromosomes 22, 6, and 11. However, consistent molecular alterations have not been identified in ependymal tumors. We studied a recurrent ependymoma in a 3-year-old patient by standard cytogenetic and molecular analysis of TP53 and MEN1 genes. In the present case, we found many of the cytogenetic features previously described as being recurrent in ependymomas, including unstable telomeric alterations. Furthermore, we detected a novel acquired heterozygous mutation in the MEN1 gene. The chromosomal instability produced by the telomeric alterations and the mutation in the MEN1 gene could be important events in the tumorigenesis of ependymomas.

Deletion of exon b3 of the BCR gene in CML: easy breakpoint mapping by a two-round PCR.

We have performed the molecular analysis for the detection of the BCR-ABL and ABL-BCR fusion genes in 50 patients with myeloproliferative disorders. All patients diagnosed with CML (13 out of 50) were positive for the BCR-ABL hybrid. Six CML patients (46%) showed ABL-BCR amplifications of the Ib-BCR type. All rearrangements but one were concordant. The aberrant case presented a deletion of exon b3, in addition to the alternative Ib-BCR and Ia-BCR. Its possible origin and relevance are briefly discussed.